Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products

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Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products Danilo D'Apolitoa,c,∗, Lucia D'Aielloa, Salvatore Pasquaa, Lucia Pecoraroc, Floriana Barberac, Bruno Douradinhab,c, Giuseppina Di Martinoc, Chiara Di Bartoloa,c, Pier Giulio Conaldic a

Unità Prodotti Cellulari (GMP), Fondazione Ri.MED c/o IRCCS-ISMETT, Via E. Tricomi 5, 90127, Palermo, Italy Unità Medicina Rigenerativa ed Immunologia, Fondazione Ri.MED c/o IRCCS-ISMETT, Via E. Tricomi 5, 90127, Palermo, Italy Unità di Medicina di Laboratorio e Biotecnologie Avanzate, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Via E. Tricomi 5, 90127, Palermo, Italy

b c

ARTICLE INFO

ABSTRACT

Keywords: Advanced therapy medicinal products (ATMP) Mycoplasmas Nucleic acid amplification technique (NAT) Validation

Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the “culture method” and “indicator cell culture method” which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

1. Introduction Advanced Therapy Medicinal Products must be controlled extensively to ensure they maintain high standards of safety, identity, purity and potency [1,2]. The safety assessment of the medicinal products includes extensive analytical tests to confirm the absence of microorganisms and of other biological and chemical impurities that might endanger the patient's health. Among them, detection of mycoplasmas is crucial, since these prokaryotic microorganisms do not possess a cell wall, making them highly resistant to antibiotics and consequently prone to cause severe pathologies, especially to persons subjected to an immunosuppression regimen [3]. European guidelines demand that samples must be screened to ensure they are mycoplasma free [4,5]. Currently, the gold standard methods recommended by European Pharmacopoeia (Ph. Eur.) are the “culture

method” and the “indicator cell culture method”. The former focusses on the detection of mycoplasma strains that can grow in specific culture media, while the latter allows for the detection of strains that do not autonomously grow in culture media, but require the presence of eukaryotic cells. Though both methods are highly sensitive, they are very laborious and time consuming [6], thus posing a problem in situations where time is a critical factor for the release and administration of the bioproduct. To overcome these problems, several alternative methods for the detection of mycoplasmas have been developed which have been shown to be more sensitive and faster than the compendial methods [6–8]. Each laboratory may develop an alternative analytical method, however it must be properly validated beforehand, to show that the results derived from it are comparable to those obtained with the gold standard methods mentioned above in terms of specificity, limit of detection and robustness [4,9]. Our

Abbreviations: GMP, Good Manufacturing Practices; ATMP, Advanced Therapy Medicinal Products; EBV, Epstein-Barr Virus ∗ Corresponding author. Unità Prodotti Cellulari (GMP), Fondazione Ri.MED c/o IRCCS ISMETT, Via Ernesto Tricomi 5, 90127, Palermo, Italy. E-mail addresses: [email protected], [email protected], [email protected] (D. D'Apolito). https://doi.org/10.1016/j.biologicals.2020.01.001 Received 29 July 2019; Received in revised form 23 December 2019; Accepted 1 January 2020 1045-1056/ © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: Danilo D'Apolito, et al., Biologicals, https://doi.org/10.1016/j.biologicals.2020.01.001

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study aimed to develop and validate an alternative method based on real time PCR that is able to identify mycoplasma species that could be contaminate cell cultures under GMP conditions, and in particular our ATMP, EBV Cytotoxic T lymphocytes (CTL-EBV) [10]. We chose a commercial kit which allows the detection of up to 90 mycoplasma spp. by targeting the hypervariable regions of the 16S rRNA gene [11]. While the indicator cell method and other nucleic acid techniques (NAT) use lower volumes, we analyzed 10 ml of matrix, the same volume used in the culture method and in an alternative method based on microarray technology [7]. The use of a higher volume allowed us to have a higher representativeness of the analytical sample, since it allows an easier detection of lower level of mycoplasma contamination. As recently suggested by several authors [12–14], the validation of an alternative NAT method must also determine the genomic copies per colony forming unit ratio (GC/CFU) of the suspensions to be analyzed. Since these methods quantify genomic material derived from both viable and dead microorganisms, assessment of the GC/CFU ratio is required to avoid an overestimation of the alternative method. Also, one may not exclude the possibility of overestimation of GC/CFU derived from cDNA amplified from bacterial RNA [12]. Thus, we determined the GC/CFU ratio of the suspensions used during the validation of our alternative protocol. Here, we describe the validation of our proposed alternative NAT method, performed as required by Ph. Eur. 2.6.7 and ICH Q2 (R1) guidelines [4,9], analyzing all requirements of specificity, detection limits, cross reaction and robustness and compared it with the results obtained with the gold standard methodologies. We were able to detect all mycoplasmas spp. in Ph. Eur. guidance, down to a threshold concentration of 10 CFU/ml while meeting all the necessary requirements established by the Ph. Eur. [4,5], showing that our method can be used as a validated alternative NAT for detection of mycoplasma contaminations in ATMP.

(Code: 1464520001. Merck KGaA, Darmstadt, Germany), harvested in the early exponential growth phase, prepared by resuspending the pellet in fresh full culture medium containing 10% CryoSure-DMSO (Code: WAK-DMSO-10. WAK-Chemie Medical GmbH, Steinbach, Germany), aliquoted and cryopreserved at −80 °C. Non-mycoplasma ATCC Gram positive bacteria, suggested by the Ph. Eur., to be used for the specificity test, were purchased from LGC Standard (Table 1). To further study the specificity of the primers of the Real Time PCR kit, we also used the following in-house nocosomial strains: Escherichia coli, Klebsiella pneumoniae and Streptococcus gordonii (Table 1), isolated from environmental sampling of hospital surfaces. Identification was performed by sequencing the 16S-rRNA as described elsewhere [15,16]. They were frozen immediately after isolation and identification, and used at passage P1, to avoid extensive passages and the possibility of occurrence of mutations. Identity and purity were confirmed throughout all the validation experiments [15,16]. Vero cells (ATCC® CCL-81™) were obtained from LGC Standards. The cells were grown at 37 °C and 5% CO2 in DMEM (Code: 12–614F. Lonza, Verviers, Belgium), with 1% L-glutamine (Code: 17–605F. Lonza) and mycoplasma free certified 10% heat-inactivated fetal bovine serum (Code: SH30070. FBS HyClone, Logan, UT, USA). 2.1.1. Titer determination For all mycoplasmas that grew in medium agar, the titer before and after freezing was determined (Table 2). The post freezing titer was evaluated in 3 different time-points (2-3 days, 3 and 4 months), to determine if stability among the frozen vials was maintained during the validation procedure. Starting from the mother suspension 12 serial decimal dilutions (ranging from 10−1 to 10−12) were prepared. For each of them an aliquot of 100 μl was plated in triplicate on Hayflick agar (Code: 1460290020. Merck KGaA). The colonies were counted after 7-9 days. The titer was determined by calculating the weighted average of the values obtained in the dilutions in which it was possible to precisely count the colonies (between 25 and 300 CFU/plate).

2. Materials and methods 2.1. Mycoplasma, non-mycoplasma strains and cell lines Reference ATCC mycoplasmas strains (Table 1) were purchased (LGC Standards, Teddington, UK) and expanded according manufacturer's instructions. Mycoplasmas were grown in Hayflick liquid media Table 1 Mycoplasma and other bacterial strains used in this work. Mycoplasma strains

Bacterial strains (specificity)

In-house nocosomial strainsa (specificity)

Acholeplasma laidlawii (ATCC® 23206™) Strain Designations: PG8 [NCTC 10116, PG8; A]

Clostridium perfrigens (ATCC® 13124™) Strain Designations: NCTC 8237 [ATCC 19408, CIP 103 409, CN 1491, NCIB 6125, NCTC 6125, S 107] Lactobacillus acidophilus (ATCC® 53544™) Strain Designation: BT1005 Streptococcus sp., group b, Type II (ATCC® 31576™) Strain Designation: MB 4055

Escherichia coli

Mycoplasma fermentans (ATCC® 19989™) Strain Designations: PG18 [G, NCTC 10117] Mycoplasma hyorhinis (ATCC® 17981™) Strain Designations: BTS -7 [ATCC 23234, D.G. ff. Edward PG 42, NCTC 10130] Mycoplasma orale (ATCC® 23714™) Strain Designations: CH 19299 [NCTC 10112] Mycoplasma pneumoniae (ATCC® 15531™) Strain Designations: FH strain of Eaton Agent [NCTC 10119] Mycoplasma arginini (ATCC® 23838™) Strain Designations: G230 [NCTC 10129] Mycoplasma hyorhinis (ATCC® 29052™) Strain Designations: DBS 1050 [3T¬6]

Klebsiella pneumoniae Streptococcus gordonii

a The in-house nocosomial strains were isolated from environmental sampling of hospital surfaces. Their identification was performed by sequencing the 16SrRNA. They were frozen immediately after isolation and identification and used at passage P1, to avoid extensive passages and the possibility of occurrence of mutations.

2

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Table 2 Titer of the mycoplasma strains suspensions before and after freezing (x107 CFU/ml).

Pre-freezing 1° Post-Freezing (2-3 days) 2° Post-Freezing (3 months) 3° Post-Freezing (4 months) Average

A. laidlawii

M. fermentans

M. orale

M. arginini

M. hyorhinis

M. pneumoniae

5,00 4,53 4,50 4,83 4,62

1,30 1,13 1,19 1,30 1,21

5,83 5,00 5,60 5,60 5,40

2,80 2,34 2,50 2,87 2,57

1,24 1,29 1,07 1,05 1,14

1,21 1,10 1,17 1,14 1,14

2.2. Mycoplasma suspensions stock for comparability study and determination of GC/CFU content

purified by ExoSap-IT (Affymetrix, Santa Clara, California, United States) and processed for sequencing using forward and reverse sequencing mix included in MicroSEQ ID System. The DNA sequencing reactions were purified using BigDye XTerminator Purification kit (Life Technologies), and sequencing was performed by capillary electrophoresis using 3500 Genetic Analyzer (Life Technologies). The sequences were analyzed using 16S rDNA 500 bp gene MicroSEQ ID Analysis software (Life Technologies).

To ensure that the comparability of the results obtained using our NAT method and the Ph. Eur. methods is correctly assessed, we prepared mycoplasma stocks of known titer, which would later be used for direct inoculations into the samples to be analyzed. Working at 4 °C and starting from the bacterial suspensions stocks described above, several 10-fold dilutions in Hayflick liquid media (Merck, Darmstadt, Germany) and 10% DMSO (WAK-Chemie Medical GmbH) were prepared and frozen for the validation of the alternative method, thus obtaining intact particles suspensions ready to use. Their titer (around 10, 100 or 1000 CFU/ml) was later confirmed, to ensure there was no viability loss during manipulation of original stocks. In order to establish a fair comparison between our proposed NAT methodology and the gold standard techniques, our mycoplasma suspension stocks were done according to GMP, to allow a high percentage of viable cells and a low degree of aggregation. Dead or aggregated cells in the stocks are thus undesirable since they may lead to an overestimation of the sensitivity of our proposed NAT method due to the detection of mycoplasma genes of not viable or aggregated bacteria [12]. The GC/CFU content of mycoplasma stocks was assessed to ensure there is no overestimation of the NAT method sensitivity during its validation. Values of GC/CFU that would differ highly among the different used microorganisms would render the accuracy of the sensitivity not realistic and very different among the different mycoplasma spp. used. To better evaluate the sensitivity of our proposed method, the GC/CFU values should be 10 or lower and not vary greatly between the different tested microorganism, so that they may be comparable between them [12]. Basically, genomic material from different suspensions was extracted with QIAsymphony SP/AS (Qiagen, Hilden, Germany) and quantified using Nanodrop system (Thermo Scientific, Waltham, MA, USA). The average content in GC/μl was determined using DNA Copy Number and Dilution Calculator software (Thermo Scientific, Waltham, MA, USA) as for developer's instructions. The GC/ml content of the suspension was calculated by multiplying the GC/μl value by the total volume of the eluate (50 μl), by 2, as the DNA was extracted from a starting sample of 500 μl, which represents half of 1 ml. Finally, for each frozen stock, quality was assessed by determining the individual GC/CFU, which gave us an average relationship between the suspension titer and its content in GC/ml. The data are summarized in Table 3.

2.4. NAT validation strategy To validate an alternative method, the Ph. Eur. suggests two different approaches. The first involves performing the new method simultaneously with the gold standard methods using the same titrated strains, while the second compares the performance of the new method results by using well titrated defined strains, thus allowing a correct comparison between experiments performed at different times. We have chosen the latter approach, using the above described suspensions of mycoplasma strains previously titrated and cryopreserved. 2.4.1. Matrix of interest To validate an analytical method, one must define previously a matrix, i.e., the sample to be tested containing all components except the one to be analyzed. When choosing a potential matrix of interest to validate our NAT, we considered the production steps of our ATMP, specific EBV Cytotoxic T lymphocytes (CTL-EBV), used to treat lymphoproliferative disorders [10]. The method involves the initial production of the lymphoblastoid cell line (LCL), by immortalizing B lymphocytes through EBV infection and subsequently produce CTLEBV, by co-cultivating T lymphocytes with irradiated EBV-LCL, the latter acting as antigen presenting cells [10]. The matrix to be used for the purpose of validation should contain all the components necessary for both the growth of LCL and CTL cells, to ensure the validation is done in a more complex matrix. Both cell types were grown in complete RPMI (Code: L0505-500. Lonza) supplemented with 10% FBS (HyClone) and 1% L-glutamine (Lonza), with the difference that LCL require the addition of cyclosporine (Code: 00078010901. Novartis, Basel, Switzerland) at a final concentration of 0,001 mg/ml, while CTL need IL-2 at a final concentration of 400 units/ ml (Code: L03A C01. Novartis). To produce the large amounts of matrix volume required for validation, we grew LCL which, due to its immortalization phenotype, allowed collection of larger volumes of culture media, as previously described [10]. For each of the 3 independent experiments, we used a matrix derived from LCL obtained from a different donor, to confirm if testing cells from different sources could affect the detection of the mycoplasmas.

2.3. Microorganisms identification The purity and identity of each microorganism (mycoplasma and non-mycoplasma strains) were determined by sequencing the 16S-rRNA throughout all the validation experiments [17]. Total genomic DNA was extracted with QIAsymphony SP/AS (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The first 500 nucleotides of bacterial 16S ribosomial RNA (rRNA) gene, encompassed 3 of 9 hyper variable regions of 16S gene, was analyzed using the MicroSEQ ID microbial identification System (Life Technologies, Carlsbad, California, United States), including PCR Master Mix, forward and reverse sequencing mix. Amplification products were detected with D1000 ScreenTape Assay using TapeStation 4200 (Agilent Technologies, Santa Clara, California, United States),

2.4.2. Culture method and indicator cell culture method The culture method was performed 3 times by 3 different operators as described elsewhere [4]. Briefly we have examined the growth properties of reference mycoplasmas strains, in Hayflick liquid and agar media (Merck, Darmstadt, Germany), in presence and in absence of the chosen matrix, evaluating if the matrix has an inhibitory effect on the mycoplasmas growth. The indicator cell culture method was performed 3 times by 3 different operators as suggested elsewhere [4]. Briefly, Vero cells were 3

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Table 3 GC/CFU content of mycoplasma suspension stocks. Strain A. laidlawii

M. fermentans

M. orale

M. arginini

M. hyorhinis

M. pneumoniae

Stock Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml Mother ≤1000 CFU/ml ≤100 CFU/ml ≤10 CFU/ml

GC/μl 3,10 × 66,9 6,21 0,61 5,50 × 46,63 4,34 0,45 3,60 × 67,25 6,21 0,62 9,10 × 37,82 3,59 0,35 8,80 × 61,26 5,55 0,54 7,90 × 71,28 6,56 0,64

GC/ml 10

5

3,10 × 6690 621 61 5,50 × 4663 434 45 3,60 × 6725 621 62 9,10 × 3781 359 35 8,80 × 6126 555 54 7,90 × 7128 656 64,4

105

106

105

105

105

Titer (CFU/ml) 7

10

107

108

107

107

107

4,62 984 92 8,9 1,21 992 94 9,3 5,40 986 90 9,1 2,57 990 96 9,4 1,14 988 95 9 1,14 990 94 9,2

6

× 10

× 107

× 107

× 107

× 107

× 107

GC/CFU 6,71 6,79 6,75 6,85 4,55 4,7 4,62 4,84 6,67 6,82 6,9 6,81 3,54 3,82 3,74 3,72 5,79 6,2 5,84 6 6,93 7,2 6,98 7

total of 24 analyses for each concentration. As indicated in Ph. Eur. [4,5], 3 independent experiments were performed using the matrix of interest, contaminated with the suspensions of mycoplasma at the 3 above referred concentrations. In parallel, since the Ph. Eur. requires a minimum level of sensitivity of ≤100 CFU of mycoplasma spp., we used two positive extraction controls with ≤100 M. hyorhinis CFU, to ensure that the minimum level of sensitivity required are respected.

infected with mycoplasma suspensions. Growth properties of mycoplasma was evaluated visualizing the specific particulate or filamentous pattern of fluorescence on the cell surface and/or in surrounding areas, using Mycoplasma Stain Kit (Sigma Aldrich, St. Louis, MO, USA) (Fig. 1). 2.4.3. Validation The detection acceptability criteria defined by the Ph. Eur. for an alternative method are 10 CFU/ml and 100 CFU/ml for the replacement of the “culture method” and of “indicator cell method”, respectively [4]. It also recommends demonstration of the following parameters: specificity, cross-reaction contamination, detection limit and robustness [4,5].

2.4.3.4. Robustness. Robustness is the ability of the method to provide reliable data even when small but deliberated variations of the usual conditions are induced, e.g., different days, users or kit manufacturer's lots. To evaluate the robustness of the method and, therefore, its ability to be unaffected by potential variations, the experiments described above for the cutoff and detection limit were performed in 3 different days, each done by a different operator, using each one a different lot of Real Time PCR kit.

2.4.3.1. Specificity. Specificity is defined as the ability to specifically discriminate the target sequence under analysis. The specificity of our method was tested by amplifying the genome of 3 bacteria that are phylogenetically related to mycoplasmas, as suggested by Ph. Eur. [4], and 3 in-house nosocomial bacterial clinical isolates (Table 1). Tests were performed by analyzing 6 samples at a concentration of 100 CFU/ ml for each microorganism. A positive detection of the amplification of these microorganisms’ nucleic acids could invalidate the specificity of our proposed NAT method. At the end, we have assessed the potential false positive rate of unrelated microorganisms to ensure the specificity of our NAT procedure.

2.5. Validation method flow chart Preliminary tests using matrix volumes of 1, 2.5 and 5 ml containing 10 or 100 CFU have shown inconsistent results (data not shown). Thus, we chose an initial volume of 10 ml to compensate genomic material loss during sample processing. The matrix was inoculated with the mycoplasma strains at a final concentration of 1, 10 and 100 CFU/ml, which were further concentrated by centrifugation at 14200 rpm for an 1 h at 4 °C. The supernatants were discarded and genomic material extracted with QIAsymphony SP/AS (Qiagen). To monitor the automatic extraction step, we used 100 μl of an inactivated Mycoplasma hyorhinis suspension with a concentration ≤1000 CFU/ml as a positive control. The addition of this standard allows to monitor the extraction step up to the minimum level of sensitivity required by Ph. Eur. This preparation was inactivated by repeated freeze/thaw cycles and with a final 5 min incubation at 80 °C, cooled to room temperature and kept at −80 °C until further use. Inactivation of M. hyorhinis suspension was confirmed by the Ph. Eur. culture method for a period of 28 days, in which no growth was observed. The feasibility of this suspension as a positive control was demonstrated by having its genomic material extracted by QIAsymphony SP/AS (Qiagen) and later submitted to amplification. Bacterial DNA was amplified using the MycoSEQ™ Kit (Applied Biosystems, Foster City, CA, USA) in a 7500 FAST Real-Time

2.4.3.2. Technical cross-contaminations. Cross-contaminations involve an undesired positive signal derived from other samples tested in parallel. To ensure our proposed NAT method and procedure associated were not prone to cross reaction contaminations, we analyzed 6 positive samples with at least 1000 CFU/ml alternated with 6 negative samples (water) in the same well plate. The evaluation involved the simultaneous handling and processing of the 12 samples (Fig. S1). 2.4.3.3. Detection limit. Detection limit refers to the lowest number of microorganisms that can be detected. To determine the positive cutoff point, i.e. the minimum number of target sequences, by sample volume, which must occur in at least 95% of the samples tested, we performed 3 independent real-time PCR runs, testing 8 repetitions of the following microbial suspensions: 100 CFU/ml, 10 CFU/ml, and 1 CFU/ml, for a 4

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not show any inhibition activity, as shown by the experiments results in which matrix was included. Results are summarized in Table 4. The indicator cell culture method was shown to have a sensitivity of 100 CFU/ml M. orale and is able to detect qualitatively the growth of M. hyorhinis. The results showed that the matrix did not inhibit the growth of M. hyorhinis and M. orale. The results are summarized in Table 5. 3.3. Validation 3.3.1. Specificity In order to further demonstrate the specificity of the commercial kit, already validated by the kit manufacturer, we analyzed the results obtained by amplifying the genome of the 3 bacteria phylogenetically correlated with mycoplasmas and 3 in-house nosocomial bacterial clinical isolates. The results showed that the primers specifically recognize the mycoplasmas genome and gave no amplification signal for any of the microorganisms tested (Fig. 3). Conversely, the PCR controls (K+PCR, provided by the kit), extraction controls (K+EXTR, M. hyorhinis suspension) and matrix plus internal control (MIC) showed the specific amplification signal expected for mycoplasma, confirming the data provided by the kit manufacturer [11]. Thus, the MycoSEQ™ Mycoplasma Detection Kit, with Discriminatory Positive Control kit (Applied Biosystems) is specific to the mycoplasma samples. 3.3.1.1. Technical cross-contaminations. As with traditional PCR, realtime PCR reactions can be affected by nucleic acid contamination, leading to false positive results. To evaluate cross-contamination between samples and contamination from laboratory equipment used during our procedure, we handled 6 positive samples with at least 1000 CFU/ml alternated with 6 negative samples (water). The results shown no amplification in the water samples (Fig. S1). Moreover, in none of the experiments performed during the validation of the method, a cross-contamination of the negative samples occurred, demonstrating that all the phases of the process, if well performed, do not lead to false positives in the negative samples, even when in the neighborhood of samples with a high content of mycoplasma particles. We should note that we used in this work only GMP grade, mycoplasma free reagents such as FBS and water, to reduce the possibility of reagent derived mycoplasma contaminations.

Fig. 1. Vero cells infected with A) M. hyorhinis and B) M. orale during the performance of the indicator cell culture method. The cells show particulate or filamentous fluorescence around the cell nuclei and in the intercellular spaces. Stained performed using Mycoplasma Stain Kit (Sigma Aldrich). Scale bar 10 μm.

3.3.2. Cutoff and detection limit determination The results obtained for the experiments regarding limit of detection and determination of cutoff are summarized in Table 6. In all 3 independent experiments and for all mycoplasmas the matrix did not exhibit interference. For all mycoplasma species the method showed a cutoff ≤10 CFU. The alternative method reaches the requirements of the Ph. Eur. with respect to sensitivity [4].

PCR (Applied Biosystems), according with the manufacturer's instructions. The scheme of the proposed method can be observed in Fig. 2. 3. Results 3.1. Titer and determination of genome copy number to CFU ratio (GC/ CFU)

3.3.3. Robustness The results obtained for robustness are shown in Table 6. The tests were performed by 3 operators in 3 different days using each one a different lot of Real Time PCR kit. All test results met the acceptance criteria for the extraction control, PCR positive control and negative control. The analysis of the results showed that the method is very robust, since the results obtained were not influenced by any variable taken into consideration.

The bacterial strains used for validation are those suggested by Ph. Eur. [4] (Table 1). For each strain, the stocks were prepared and frozen for later use and their titer was determined (Table 2). The titers were shown to be stable through the length of time in which the validation tests were performed. The number of genomic copies (GC/ml) was determined for all species of Mycoplasma to be tested by the proposed NAT method. As shown in Table 3, all our frozen Mycoplasma stocks have a low GC/CFU value (from 3,54 to 7,2), which indicates that our strains are optimal standards for the evaluation and comparison of the alternative NAT method with official methods.

4. Discussion The gold standard methods recommended by the Ph. Eur. for the detection of mycoplasma contamination in the samples of pharmaceutical preparations, are the culture method and the indicator cell culture method [4,5]. Although these methods are effective and sensitive, they are very laborious and time consuming, and their results are only available after several days (up to 28 days). This last aspect is very critical, especially for those ATMP which require a rapid release for immediate administration to the patient.

3.2. Culture and indicator cell culture methods The culture method was shown to have a sensitivity of 100 CFU/ml for the 4 tested mycoplasmas indicated by Ph. Eur. [4]. The matrix did 5

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Fig. 2. Scheme of the proposed NAT method for mycoplasma detection in ATMP. Table 4 Detection of mycoplasma strains using the culture method, in the presence or absence of matrix.a.

A. laidlawii w/o Matrix with Matrix M. hyorhinis w/o Matrix with Matrix M. orale w/o Matrix with Matrix M. pneumoniae w/o Matrix with Matrix

100 CFU/ml

10 CFU/ml

Liquid

Solid

Liquid

Solid

Liquid

Solid

100% 100%

100% 100%

0% 0%

11,1% 0%

0% 0%

0% 0%

100% 100%

100% 100%

0% 0%

0% 0%

0% 0%

0% 0%

100% 100%

100% 100%

0% 0%

0% 0%

0% 0%

0% 0%

100% 100%

100% 100%

0% 0%

0% 0%

0% 0%

0% 0%

Table 5 Detection of mycoplasma strains using the indicator cell culture method, in the presence or absence of matrix.a.

1 CFU/ml

M. orale w/o Matrix with Matrix M. hyorhinis w/o Matrix with Matrix a

≤ 1000 CFU/ml

≤ 100 CFU/ml

≤ 10 CFU/ml

100% 100%

100% 100%

0% 0%

Qualitative Growth 100% 100%

Results shown are derived from 18 replicates (6 replicates per experiment).

the following aspects: sensitivity, limit of detection and cutoff, crossreaction and robustness. This study reports the validation strategy of an alternative method we developed for detection of mycoplasma DNA using the commercial kit MycoSEQ™ Mycoplasma Detection Kit. This kit has been used previously in evaluation of the feasibility of NAT assays for mycoplasma detection [18,19], showing low risk of cross contaminations and efficiently detecting several mycoplasmas spp. However, validation of an alternative NAT protocol for mycoplasma detection in agreement with the Ph. Eur. standards using the MycoSEQ™ Mycoplasma Detection Kit has never been reported, to the best of our knowledge. The compendial methods are also used in the USA [18] and Japan [6]. The USP foresees general alternative procedures for

a Results shown are derived from 18 replicates (6 replicates per experiment). Results are expressed in percentage of bottles which displayed mycoplasma growth e.g., 100% means that growth of mycoplasma was observed in all 18 replicates.

The Ph. Eur. gives the possibility to use an alternative analytical method, provided that it gives results which are, at least comparable, or better to those of the compendial methods, concerning the evaluation of 6

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Fig. 3. Specificity test for validation of the proposed NAT method. The genome of bacteria that are phylogenetically related to mycoplasmas or derived from in-house nosocomial clinical isolates were analyzed using the MycoSEQ™ Mycoplasma Detection Kit, each sample being at a concentration of 100 CFU/ml. As expected, none of these samples showed amplification signal. The PCR control (K+PCR), extraction control (K+EXTR, M. hyorhinis suspension) and matrix plus kit internal control (MIC) displayed specific amplification signals, as expected.

as stable titer over time. Subsequently, we produced the mycoplasmas stocks to be used for the comparability study and determined their titers. Due to the fact it is based on a PCR strategy, the genomic material of either living and dead bacterial cells as well as of cellular aggregates may be accounted for in the proposed alternative method, which might affect the evaluation of its sensitivity, by over-estimating or under-estimating the amount of mycoplasma present in the samples. As mentioned above, to overcome this problem, we assessed the quality of the frozen stocks, determining the GC/CFU ratio. This index correlates the quantity of total DNA with its source and, the smaller this value is, the less DNA from dead cells is present in solution [12]. Thus, the GC/CFU ratio provides a valuable information on the quality of stocks and their suitability for assessing the sensitivity of an alternative method based on the detection of nucleic acids. Our results, summarized in Table 3, showed that our frozen suspensions have a low GC/CFU ratio (from 3,54 to 7,2), therefore being excellent standards for the NAT method validation, as required by the Ph. Eur. and reported by other research groups [12,13]. The Ph. Eur. recommends as standards for validation the following mycoplasma spp.: A. laidlawii, M. fermentas, M. hyorhinis, M. orale, M. pneumoniae or Mycoplasma gallisepticum, Mycoplasma synoviae (for avian derived materials), M. arginini and Spiroplasma citri (for insect or plant derived materials) [4]. Specificity must be demonstrated using bacterial spp. other than mycoplasma, such as Clostridium, Lactobacillus and

Table 6 Determination of the cutoff of the proposed validation NAT method. Mycoplasma detection (total positive signal/total reactions performed)

A. laidlawii M. hyorhinis M. orale M. pneumoniae M. fermentans M. arginini

100 CFU

10 CFU

1 CFU

24/24 24/24 24/24 24/24 24/24 24/24

24/24 23/24 24/24 24/24 24/24 23/24

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mycoplasma detection as long as they are validated and provide the same or better results than the gold standard methods [6]. The international pharmacopeias are currently working to align their strategies, which would imply that alternative NAT protocols for mycoplasma detection would be worldwide accepted, thus the technique proposed by us could also be used in other countries other than European ones. Initially we produced stocks of the Mycoplasmas spp. recommended by the Ph. Eur. (Table 1) and evaluated the titer along the validation period (Table 2). The results showed that our method of production, freezing of mycoplasmas and the choice of GMP grade products to grow and freeze microorganisms, allowed frozen suspensions to be maintained 7

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Streptococcus, due to their close phylogenetic relation with mycoplasma spp. [4]. As reference standards for validation of a NAT alternative method to the gold standard methods, one must use as reference standards copies of mycoplasma nucleic acid, since it is not possible to use directly growing organisms, such as bacterial CFU. Thus, the relationship between CFU and mycoplasma genomic copies must be established beforehand. Different methods could be used to measure GC value, i.e. UV based method or qPCR based methods. For GC determination by qPCR, references DNA are indispensable to create the standard universal curve (e.g., plasmid DNA with the insertion of the targeted genomic region, etc) and their characteristics could cause variation in the GC assessment. If the standard curve contains only information about the 16S rRNA gene, the GC calculation must take in consideration the real number of gene copies present in the sample and adjust it to the number of 16S rRNA genes of the chosen reference mycoplasma spp. DNA, since some mycoplasmas spp. contain two 16S rRNA genes. Different genomic DNAs corresponding to the test spp. could be used to overcome this issue [12]. In order to validate our protocol, we use the suspensions of mycoplasma strains previously titrated and cryopreserved, to perform the two golden standard Ph. Eur. methods [4,5] and the in-house Real-Time PCR method. The results showed that the matrix chosen does not exhibit interfering activity. In fact, the experiments performed with and without the matrix have the same robust results of growth with a methodological sensitivity of ≤100 CFU/ml, i.e., less than 100 CFU were detected in 1 ml of sample, which is the minimum level of sensitivity established by the Ph. Eur. [4]. Subsequently, we designed our validation strategy taking into account our specific process and the instrumental equipment of our Quality Control laboratory. One of the key issues to keep the whole process under control is the definition of a standard that can be used as an extraction control to be sure that the extraction step works correctly, both in the validation phase and during routine analyses [18]. This control will guarantee that the negative amplification signal is due to the absence of mycoplasma and not to a false negative of the extraction step. The results obtained during the validation have shown that the M. hyorhinis standard we developed is suitable for controlling the critical step of extraction, up to the minimum level of sensitivity required by Ph. Eur. Before starting the validation of the method, we tested the possibility of cross reaction, by processing in parallel and simultaneously highly contaminated samples (≥1000 CFU/ml) and samples containing only water. Our results showed only positive signals for the analyzed contaminated samples but none for those containing water, demonstrating that cross reactions are not prone to occur using our method. Next, we tested the specificity of the kit, to ensure its primers do not amplify the genetic material of phylogenetically related organisms. We observed that the primers of the kit fail to amplify the genomes of mycoplasma phylogenetically related organisms and of other nosocomial clinical isolates obtained in-house (Table 1). This is important, as other commercial kits for mycoplasma detection have shown to amplify unspecific genetic material of unrelated bacterial spp. [20]. Conversely, the kit used in our validation did not exhibit cross detection when the genomes of reference strains of Streptococcus sp. Group 2, Clostridium perfringens and Lactobacillus acidophilus or from nosocomial clinical isolates of E. coli, S. gordonii and K. pneumoniae were subjected to amplification. To increase the representability of the analytical sample, we processed a matrix volume of 10 ml. The same volume was successfully used by another validation process, although their alternative method focused on microarray technology [7]. Using this volume, the analysis of matrix samples contaminated with suspensions with a known titer of mycoplasmas has shown, throughout the methodological process, highly reproducible and robust results. The comparative analysis of the results obtained with the NAT method and those obtained with the compendial methods has shown that, with the former, robust and reproducible results are obtained with a sensitivity towards all mycoplasmas tested of ≤10 CFU, as required by the Ph. Eur. [4,5].

In addition, but not less important, as required by the CFR 21 part 11 regulations [21] and by the Annex 11 of the Volume 4 of Good Manufacturing Practice Medicinal Products for Human and Veterinary Use [22], all the operations performed by the operator using the Applied Biosystems® 7500 fast Real-Time PCR system tool are recorded, ensuring the incorruptibility of the data and allowing audit trails, which represent a fundamental aspect of the GMP Quality Control activities. To validate our NAT suggested procedure, we have chosen as matrix one of our ATMP, EBV-CTL growth media. However, we do not anticipate any problem in applying this protocol to any other similar matrix to assess their levels of mycoplasma contamination. For those interested in applying our protocol, although we have used M. hyorhinis we note that any mycoplasma spp. can be used as extraction control. All other controls are present in the MycoSEQ™ Mycoplasma Detection Kit and should be used as in agreement with the manufacturer's instructions [11]. During routine checks on the method sensitivity, we suggest the use of reference standards at the concentrations recommended by the Ph. Eur., to ensure the sensitivity of the proposed method is respected. This means that the limits of detection must be inferior to 10 CFU/ml or to 100 CFU/ml if our proposed method is used to replace the culture method or the indicator cell culture method, respectively [4]. When using different matrixes or the same matrix with different components or when establishing this protocol in a new laboratory, it is always advisable to repeat the validation step to demonstrate that those changes do not affect the efficiency of the mycoplasma detection [2,23]. Also, as a good laboratory practice, it is advisable to train new operators unfamiliar with the protocol in order to confirm if they have the required proficiency to obtain similar results to those previously validated. 5. Conclusion In conclusion, the results obtained using the commercial kit MycoSEQ™ Mycoplasma Detection Kit together with the QIAsymphony SP/AS automatic extraction system and the Applied Biosystems® 7500 fast Real-Time PCR system tool showed that the kit is suitable for ATMP production processes quality control methodologies, ensuring safe, reliable and robust results in less than 1 day and allowing for controls in critical process steps, such as genomic extraction and Real Time PCR. Declaration of competing interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We gratefully acknowledge Nicola Cuscino for the management of the instruments used for this validation, Dr. Monica Miele, Dr. Mariangela Di Bella, Dr. Francesca Timoneri for technical assistance with cellular assays and matrix production, and Silvana Lo Giudice for technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biologicals.2020.01.001. References [1] Use C for MP for H. Guideline on human-cell-based medicinal products. United Kingdom: European Medicines Agency; 2006. [2] Parliament E. Regulation 1394/2007 of REGULATION (EC) No 1394/2007 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004. European Union; 2007.

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